The biosynthesis of the steroid precursor molecule lanosterol is a remarkable process in which the enzyme-bound substrate 2,3-S-oxidosqualene forms four new carbocyclic rings by a cascade of cation-alkene addition reactions, followed by a series of 1,2-methyl and hydride shifts. The work presented in this thesis is a computational study of the reactions of compounds designed to model the oxidosqualene-lanosterol cyclisation in order to establish details of the mechanism of this amazing cyclisation. The initiation of oxidosqualene cyclisation has been modelled by the intermolecular reaction of protonated oxirane and methylpropene. The SN2-like ring opening of the protonated epoxide is strongly exothermic with a low barrier to reaction; the geometry of the gas phase reaction has been found to be significantly affected by hyperconjugative stabilisations and low energy steric interactions. The energy profile and geometry of this reaction can now be compared to analogous intramolecular reactions such as the formation of the lanosterol A-ring. The competing five- and six-membered cyclisations of a series of substituted A-ring model compounds was investigated. It has been found that the facile cleavage of the protonated epoxide causes the reaction to behave more as an electrophilic addition than as a nucleophilic ring-opening substitution. This behaviour accounts for the general preference of protonated epoxides to react at the more substituted carbon atom, while epoxides in neutral or basic media react at the least sterically hindered carbon. With consideration for Baldwin's rules for ring closure, it is seen that the series of model compounds generally favours six-membered ring formation endo at the epoxide. The formation of the lanosterol B-ring was studied using a bicyclic model system. Previous computational studies had predicted the B-ring to close with readily with an activation energy of less than 1 kcal mol-1, however the present study has found a significant barrier to cyclisation of ca. 5-7 kcal mol-1 in this gas-phase model at the HF/6-31G(d) level of theory. This barrier is thought to arise from the closure of the B-ring in a sterically hindered twist-boat conformation.

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.

The design of new, improved NO-donor drugs is an important pharmacological objective due to the biological importance of nitric oxide. N-Hydroxyguanidines represent a useful class of NO donors where the mechanism of action is based on the biosynthetic pathway for NO. Thirty new N-arylalkyl-N’-hydroxyguanidines were synthesized and their vasodilatation activity examined by myography in rat aortic rings. The observed relaxations were reversed by ODQ, which is an inhibitor of the guanylate cyclase, implying that this was an NO dependent vasodilatation. The most active compounds were also tested in the isolated perfused kidney (IPK) giving the vasodilatation properties. Preliminary results indicated that N-phenyl-N’- hydroxyguanidine showed the best pharmacological profile with EC₅₀= 19.9 μM and ca. 100% reversibility with ODQ. A series of N-phenylalkyl-N’-hydroxyguanidines were synthesised. NO donor activity was found to be fairly constant up to three methylene groups, and then decreased. Substitutions in the benzene ring of N-phenylethyl-N’-hydroxyguanidine demonstrated that various electron-withdrawing and electron-donating groups in the para position did not significantly affect the NO donor activity of this series of analogues. The nitro and trifluoromethyl substituted compounds gave the best biological profiles. Additionally, a novel heterocyclic, N–furfuryl-N’–hydroxyguanidine possessed very promising vasodilatation properties. In general, almost all the N-arylalkyl-N’-hydroxyguanidines behaved as potent NO donors in the rat aorta assay. In order to establish the influence of the free NH₂ group in the hydroxyguanidine functionality on the vasodilatation properties, N,N-dimethyl and N-methyl-N’- hydroxyguanidines were successfully synthesised. Unfortunately, they have not been tested yet in the biological assay. However, their NMR spectra showed some unusual features and their detailed analysis and X-ray data are presented herein. In addition a series of hydroxamic acids was synthesised and the NO donor activity investigated using the same biological methodology. It was found that the 3-phenylpropionohydroxamic acid was the most potent compound with EC₅₀ = 6 μM and ODQ = 96%. However, behavior in the IPK indicated that hydroxamic acids did not undergo the same biological pathway as in the rat aorta. Two different types of enzyme-activated pro-drugs were designed using N-hydroxyguanidines as the NO donating molecule. Synthetic studies towards these targets were carried out using various synthetic approaches. The desired molecules have not yet been synthesised but the chemistry explored so far has indicated potentially more successful approaches that could be attempted.

<p>The molybdenum cofactor (Moco) is an essential enzyme cofactor found in all kingdoms of life. Moco plays central roles in many vital biological processes, and must be biosynthesized de novo. During its biosynthesis, the characteristic pyranopterin ring of Moco is constructed by a complex rearrangement of guanosine 5'-­triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC. However, the mechanisms and the functions of the two enzymes are under significant debate. To elucidate their physiological roles, I took a multidisciplinary approach to functionally characterize MoaA and MoaC in vivo and in vitro. In this dissertation, I report the first isolation and characterization of the physiological MoaC substrate, 3',8-­ cyclo-­7,8-­dihydro-­guanosine 5'-triphosphate (3',8-cH2GTP). I also report the first X-­ray crystal structures of MoaC in complex with this highly air sensitive substrate, and its product cPMP. These studies, combined with in vitro experiments using substrate analogs, catalytically impaired mutants, and synthetic peptides, have enabled me to delineate the functions of the Moco biosynthetic enzymes, MoaA and MoaC, and proposed mechanistic models for their roles in the formation of cPMP.</p> / Dissertation

Polyketides are a group of bioactive natural products synthesized by bacteria, fungi and plants with various acyl-CoA precursors, such as malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA. A sufficient supply of these precursors is a prerequisite for the high level production of polyketide products. A thorough understanding of relative roles of various metabolic pathways involved in precursor supply makes increased production by genetical manipulation, and thus rational strain improvement, a reality. Monensin A is a polyketide antibiotic assembled from one ethylmalonyl-CoA, seven methylmalonyl-CoA and five malonyl-CoA molecules by Streptomyces cinnamonensis. In the present work, the origin of these biosynthetic precursors was investigated using an industrially mutagenized monensin producer and industrial fermentation conditions. A hitherto disregarded metabolic pathway was discovered to play a significant role in providing methylmalonyl-CoA for monensin biosynthesis by gene disruption, isotope-labeling of monensin and analysis of in vivo acyl-CoA pools. This pathway starts from biosynthesis of butyryl-CoA from two molecules of acetyl-CoA, and goes through the intermediate of isobutyryl-CoA, and finally produces methylmalonyl-CoA by direct oxidation of the pro-S methyl group of isobutyryl-CoA.Industrial fermentation of the industrially mutagenized monensin producer yields significantly more monensin than the routine laboratory fermentation. This suggested the presence of abundant in vivo malonyl-CoA and methylmalonyl-CoA in this process and presented an opportunity to utilize it as a biological system for the high-titer production of heterologous polyketides derived from malonyl-CoA and/or methylmalonyl-CoA. The tetracenomycin C polyketide synthase (PKS) synthesizes tetracenomycin C, a polyketide with ten molecules of malonyl-CoA. In this work, the tetracenomycin C PKS gene cluster was introduced into two industrially mutagenized strains of Streptomyces cinnamonensis. Unprecedented multi-gram/liter of tetracenomycin production was observed in the resulting two strains, indicating the high potential of industrially mutagenized monensin production strains as efficient hosts for the production of malonyl-CoA-derived polyketides. For additional improvement in tetracenomycin yield, we attempted to increase malonyl-CoA supply to tetracenomycin C PKS by genetically manipulating metabolic pathways affecting production of malonyl-CoA and eliminating competition from monensin PKS for malonyl-CoA. However, only decreased tetracenomycin production was observed, demonstrating that the regulation of malonyl-CoA-related metabolic pathways is a complex process.

Tuberculosis emerged from its grave to be one of the deadliest diseases of the present time after recently developing a synergy with AIDS. A fatty acid condensing enzyme-mtFabH has been proposed to connect the key processes involved in biosynthesis of mycolic acids, an important component of mycobacterial cell wall. It condenses long acyl Coenzymes A (CoA; up to C20CoA) with malonyl Acyl Carrier Protein (ACP) to form the elongated β-ketoacyl-ACP which further undergoes rounds of elongation to form mero-mycolate branch of mature mycolic acids. Owing to its proposed central position in mycolic acid synthesis, mtFabH has attracted considerable attention as a good anti-mycobacterial target.In this study, we utilized important biochemical tools such as site directed mutagenesis, mass spectrometry and X-ray crystallography to address some of the key unanswered questions regarding the intricate workings of mtFabH. We solved the first co-crystal structure of substrate C12CoA with mtFabH and further analyzed the substrate specificity of this acylation step. This structure depicts the mode of acyl-CoA binding in mtFabH channels; and its comparison with the parallel E.Coli-acetyl CoA structure provides important similarities and differences in substrate binding in these two FabH enzymes. It also posed an important question about the trajectory of long acyl chain CoA into the deep and "seemingly closed" substrate binding pocket of mtFabH. By utilizing disulfide-based inhibitors, we showed that large conformational changes are necessary to facilitate ligand trafficking in mtFabH while the high catalytic turnover rate of the enzyme is maintained. We also proposed the most likely location of the involved loop.A much faster and less cumbersome assay for mtFabH was also developed and it was utilized to characterize a series of inhibitors. This assay utilizes the commercially available radioactive malonyl-CoA in lieu of malonyl-ACP, the physiological substrate, and thus can serve as ACP independent assay for mtFabH.These studies further our understanding of the biochemistry of mtFabH, which along with the faster assay could be helpful in designing potent mtFabH inhibitors as anti-tubercular agents in the future.

Phosphoglucosamine mutase (GlmM) is an enzyme of bacterial cell wall biosynthesis. The main aim of this thesis was to find out, whether gene glmM is essential for viability of Streptococcus pneumoniae. Therefore, we prepared merodiploid strain containing two copies of glmM; the genomic gene and ectopic copy under control of zinc inducible promoter. Subsequently, depletion strain was prepared by deletion of genomic copy of glmM. This strain was further used for analysis of viability and phenotype features in the medium containing various concentrations of zinc ions, an inducer of ectopic glmM expression. We found out, that the viability of this strain was strictly dependent on the concentration of inducer and further, that depletion of GlmM resulted in remarkable morphological defects. The rescue of mutant strain was observed after addition of inducer up to the level of the control sample. These results have provided the evidence of glmM essentiality for S. pneumoniae viability. Furthermore, we analyzed, whether phosphorylation of key amino acid residues, S99 and S101, is essential for GlmM functionality. Four different strains were prepared by means of site-directed mutagenesis expressing glmM with substitutions of key serine residues for alanine or glutamic acid. Since deletion of chromosomal locus in...

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